Lysyl Oxidase Activity Research Articles

Effect of dietary cadmium and/or copper on the bone lysyl oxidase in copper-deficient rats relative to the metabolism of copper in the bone

Effects of cadmium (Cd) on lysyl oxidase activity and copper (Cu) metabolism in bone were studied using Cu-deficient rats supplemented with Cu and/or Cd in a diet. When fed for 8 weeks on a diet containing 0.3 ppm or less Cu (-Cu diet), weanling rats revealed anemia, and markedly decreased plasma ceruloplasmin activity and serum Cu to less than 15% of normal level, showing features of Cu-deficiency. These rats were divided into four groups and refed for another 2 weeks on the following diets: Group I, -Cu diet; Group II, -Cu diet with 50 ppm Cd (+Cd diet); Group III, -Cu diet supplemented with 15 ppm Cu (+Cu diet); group IV, -Cu diet with both Cu and Cd (+Cu/+Cd diet). After 2 weeks, serum Cu levels of Groups I, II, III and IV were 1.8, 0.8, 78 and 74% of the normal control level (1.438 +/- 0.060 micrograms/ml), respectively. Concentrations of Cu in epi- and metaphyses of the control group, Groups I, II, III and IV were 1.45 +/- 0.20, 0.67 +/- 0.08, 0.76 +/- 0.12, 1.40 +/- 0.31 and 1.22 +/- 0.05 micrograms/g wet tissue, in that order. Concentrations of Cd in epi- and metaphysis increased in only Groups II and IV and were 0.15 +/- 0.03 and 0.18 +/- 0.01 micrograms/g wet tissue, respectively. Thus, having both Cd and Cu supplements in a diet did not inhibit each other's uptake into the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of lysyl oxidase in experimental hepatic fibrosis

The synthesis of lysyl oxidase, which initiates the cross-linking of collagen and elastin, was investigated in carbon tetrachloride (CCl4) induced fibrotic liver of rat. Lysyl oxidase activity of the fibrotic liver was 4 times greater than that of normal liver. mRNAs from the livers of normal and CCl4-treated rats were prepared for in vitro protein synthesis and the products were analyzed by immunoprecipitation with a monoclonal antibody against lysyl oxidase. The mRNAs from the fibrotic liver gave more than 3 times higher level of messenger copies for lysyl oxidase than did mRNAs from normal liver. The molecular weight of the nascent lysyl oxidase was 48,000.

Correlations between age and rat dermis modifications. Ultrastructural-morphometric evaluations and lysyl oxidase activity.

The extracellular matrix is a complex, integrated macromolecular system which plays a crucial role in the economy of each organ. In this study we focused our attention on the correlations between age and rat skin dermis. The latter was chosen as a model of the connective tissue, and was analyzed by means of electron microscopy and by measurement of the activity of lysyl oxidase, the enzyme involved in collagen and elastin crosslink formation. Ultrastructural and morphometric evaluations associated to body weight growth, showed a progressive increase in the amounts of extracellular components and a progressive reduction in the cell density. Skin from adult animals appeared characterized by a well organized matrix; by contrast, in old rats, we observed several degenerative features such as the disorganization of collagen bundles, the vacuolization of elastic fibers, and the atrophy of the mesenchimal cells. Morphometric evaluations in old animals showed a slight but significant reduction in the percentage of the total collagen measured, a fair stability in the area occupied by the elastin fibers, and an increase of the apparently non-structured matrix. The fact that lysyl oxidase activity was diminished in old rats does not corroborate the observation by several authors that increased collagen insolubility is a consequence of higher intra- and intermolecular crosslinking. This would suggest that other chemical modifications, such as crosslink oxidation or non enzymatic glycosylation, might be involved during the aging of connective tissue. The qualitative and quantitative modifications observed at all ages illustrate the correlation between connective tissue modifications and structural and/or functional properties of the skin.

Serum lysyl oxidase activity in patients with various liver diseases.

Serum lysyl oxidase activity was examined in patients with various liver diseases. The activity of the enzyme was detected mainly in the serum fraction of the supernatant 80% saturated with (NH4)2SO4, and its molecular weight was estimated to be about 30,000 by Sephadex G-150 column filtration. Mean serum lysyl oxidase activity in 18 healthy controls was 129 +/- 50 (+/- SEM) cpm/ml and was significantly increased in patients with acute hepatitis, chronic active hepatitis, alcoholic liver disease and primary biliary cirrhosis, but not in those with chronic inactive hepatitis or liver cirrhosis. Serum lysyl oxidase activity was not correlated with the histological grade of hepatic fibrosis, but appeared to reflect active hepatic fibrogenesis in patients with liver diseases.

Sudden infant death syndrome: Congenital copper deficiency

A previous paper(1) describes specific respiratory toxins likely to be associated with Sudden Infant Death Syndrome (SIDS). This paper describes ultrastructural abnormalities in the lung and other tissues in congenital copper deficiency(2). Congenital copper deficiency is associated with tissue anoxia defects in the development of myelin through a failure of phospholipid synthesis. Phospholipid is part of the membrane structure of cells. The paucity of elastin is attributed to a loss of lysyl oxidase activity. Hypoventilation is considered a feature of SIDS.

Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

TISSUE LYSYL OXIDASE ACTIVITY IS DECREASED IN MENKES' DISEASE

The X-linked recessively inherited disorder, Menkes' disease, is characterised by progressive cerebral degeneration and connective tissue abnormalities. Although the basic defect remains unknown, it appears to render copper unavailable to copper-dependent enzymes. The connective tissue defects are thus explicable on the basis of reduced activity of lysyl oxidase, which initiates the crosslinking of collagen and elastin. Since tissue levels of this enzyme have not previously been determined in Menkes' disease, we have measured its activity in extracts of skin and aorta from a patient who died at 11 months of age with unusually marked connective tissue involvement, i.e. osteoporosis, Wormian bones, pathological fractures, cutis laxa, bladder diverticula and tortuosity of arteries. We found it, indeed, to be only 10-15% of that in extracts from age-matched controls. Despite this, the ratio of β to α components of pepsin-extracted type I collagen from these two tissues appeared to be normal, and it may be that low levels of lysyl oxidase affect the crosslinking of elastin to a greater extent than that of collagen.

Stimulation of lysyl oxidase (EC 1.4.3.13) activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth-muscle cells.

Previous studies have indicated a greater incidence of atherosclerotic cardiovascular disease in men than in women of child-bearing age, suggesting that vascular interactions with sex steroids may effect pathogenesis in these cases. In the present study, it was found that the presence of 10-100 nM-testosterone in the growth medium of calf aortic smooth-muscle cells in culture stimulates lysyl oxidase activity approx. 2.5-fold in the medium and 5.5-fold in the fraction bound to the cell layer. Androgen receptors were identified in these cultured smooth-muscle cells, and their properties were very similar to those in the cytosolic fraction of whole bovine aortic tissue. These receptors appeared to be specific for androgen, of high affinity (Kd = 0.4 nM) and of low capacity (9000 sites/cell). The present results indicate that the aortic smooth-muscle cell is a cellular target for androgens, and thus raise the possibility that the development of fibrotic arterial lesions involving the deposition of excess collagen may in part be regulated by androgen-mediated stimulation of collagen cross-linkage formation as catalysed by lysyl oxidase.

β-アミノプロピオニトリルによるラット解離性大動脈瘤へのビタミンB<sub>6</sub>の関与

It is well known that pyridoxal phosphate (PAL-P) works as a cofactor of lysyl oxidase, an enzyme that is absolutely necessary to form the crosslinking in collagen and elastin and β-aminopropionitrile (BAPN) produces dissecting aneurysm in aorta of rats and another animals, because it inhibits the activity of lysyl oxidase. But the direct relationship between PAL-P and BAPN is unknown, thus authors studied physiological effects of PAL-P on enzymatic reactions of cross-linking formation.In this study, authors observed that PAL-P combined with BAPN and considerably inhibited the depression of activities of vitamin B6 dependent enzymes by BAPN in vitro. In addition, allylamine and cysteamine that have been reported as toxic agents for vascular wall, also inhibited the activities of vitamin B6 dependent enzymes as same as BAPN and INH.From the above results, it was assumed that BAPN inhibited the lysyl oxidase by binding with PAL-P that is necessary for the activation of lysyl oxidase.

Collagen metabolism in fibrotic liver. Effects of concanavalin A and aggregated myeloma immunoglobin G.

We previously have shown [Takahashi & Kobayashi (1982) Hepatology 2, 249-254] that the administration of concanavalin A to mice with schistosomiasis caused liver collagen content to be reduced by 50%. Here we report the effects of concanavalin A and aggregated mouse myeloma IgG on liver lysyl oxidase activity and present further evidence concerning the possible mechanism by which the liver collagen content was decreased in infected-treated mice. The lysyl oxidase activity at 8 weeks after infection in both treated mice and untreated infected controls was about 28-fold greater than in the age-matched uninfected controls. The specific radioactivity of intracellular free [14C]proline, the rate of collagen synthesis, the ratio of collagenase-sensitive, protein-bound, hydroxyproline to proline of collagen and the intracellular degradation of newly synthesized collagen were similar in treated animals and in untreated infected controls. In contrast, the extracellular degradation of newly secreted collagen and the specific radioactivity of protein-bound [14C]hydroxyproline in the agent-treated groups were about 2-fold greater than those in the untreated infected controls. These results suggest that the observed 50% decrease in content of liver collagen of mice treated with the agents apparently was due to the increased extracellular degradation of newly secreted collagen.

家兎口蓋粘膜創傷治癒時のリシルオキシダーゼ活性と病理組織所見について

The purpose of this paper is to investigate the mechanism of scar formation on the palate which can inhibit maxillary development after cleft palate surgery. After 4 types of operations (elevation of the mucoperiosteum, excision of the mucosa, excision of the periosteum, and excision of the mucoperiosteum) were carried out on rabbit palates, the healing processs was studied histologically and biochemically by measuring lysyl oxidase activity which is necessary for collagen cross-linking.The results were as follows.The lysyl oxidase activity increased sharply and peaked at 4 days after elevation of the mucoperiosteum or excision of the mucosa when fibroblast had histologically appeared in the wound.On the other hand, the activity increased gradually up to 14 days after periosteum excision, or excision of the mucoperiosteum, showing histologically remodeling of the underlying bone.The results suggest that it is very important to avoid periosteum injury and to decrease the wound area exposing the bone without the mucoperiosteum as much as possible for growth of the maxilla after cleft palate surgery.

β-アミノプロピオニトリルによるラット解離性大動脈瘤へのビタミンB<sub>6</sub>の関与(第2報)

Physiological effects of pyridoxal 5'-phosphate (PLP) on enzymatic reactions of cross-linking formation in collagen and elastin were studied. In the previous report of authors, it was observed that PLP combined with β-aminopropionitrile (BAPN) and considerably inhibited the BAPN induced depression of activities of vitamin B6 dependent enzymes in vitro. In this study, we performed an animal experiment using SD rats fed the lathyrogen (BAPN) and the effects of PLP on these animals were examined.Thirty male SD rats were separated the following five groups: Group 1, three rats fed just a standard diet as control. Group 2, six rats receiving 250mg/kg/day of pyridoxine hydrochloride (PIN). Group 3, seven rats receiving 300mg/kg/day of BAPN. Group 4, seven rats receiving the same dose of BAPN as rats of group 3 and 125mg/kg/day of PIN. Group 5, seven rats receiving the same dose of BAPN as rats of group 3 and 250mg/kg/day of PIN. Authors observed the occurrence of death to be due to lethargic dissection of aorta for 189 days.During the observation period, six rats of group 3 and six rats of group 4 died evidently on account of toxic effects of BAPN. But there were three survivors in group 5. It was generally observed that mean value of days required to occur lethargic dissection of aorta in group 5 was longer than those of groups 3 and 4.From these results, it was concluded that the existence of sufficient dose of vitamin B6 may be necessary for mainteining the activity of lysyl oxidase which is the key enzyme for cross-linking formation of collagen and elastin, and lethargic dissections of aorta in rats caused with BAPN were inhibited by the administration of vitamin B6.

Effect of Vitamin B6 on Dissecting Aneurysm of Rats Fed β-Aminopropionitrle

Effects of pyridoxal 5′-phosphate on enzymatic reactions of cross-link formation in collagen and elastin were studied. In this study, we observed that pyridoxal 5′-phosphate combined with β-aminopropionitrile to competitively reverse the inhibition of activities of vitamin B6-dependent enzymes brought about by β-aminopropionitrile in vitro. In addition, we observed that a sufficient dose of pyridoxine hydrochloride prevented the occurrence of dissecting aneurysm of SD rats fed β-aminopropionitrile. From the above results, we assume that β-aminopropionitrile inhibited the lysyl oxidase by binding with the pyridoxal 5′-phosphate that is necessary for the activation of lysyl oxidase.

Secretion of lysyl oxidase by cultured human skin fibroblasts and effects of monensin, nigericin, tunicamycin and colchicine

Lysyl oxidase is an extracellular enzyme that initiates crosslink formation in the major connective tissue proteins, the collagens and elastin. This enzyme activity accumulated in a fresh medium of cultured human skin fibroblasts for at least 24 h, but the accumulation was distinctly non-linear after the first 12 h. Most of the total enzyme activity was present in the medium, the activity found in the cell layer representing about 30% of the total activity at 4 h, and about 10–15% at 24 h. The bulk of the cell-layer-associated activity appeared to be extracellular, as more than half was lost upon trypsinization. Culturing of the cells for 8 h in the presence of either monensin or nigericin, ionophores known to inhibit the secretion of many proteins at the level of the Golgi complex, markedly reduced the accumulation of lysyl oxidase activity in the medium. Monensin was particularly effective, as it produced a distinct inhibition even at a 10 nM concentration, reaching 50% at 30 nM. Both ionophores also reduced enzyme activity in the cell layer, whereas no definite decrease was seen in the activity of the trypsinized cells. The effect of monensin was evidently not due to any general toxicity on the part of the drug, since even a 500 nM concentration gave no inhibition of the incorporation of [ 3H]leucine into total protein. Tunicamycin also reduced lysyl oxidase activity in the medium and to a lesser extent in the cell layer, but the effective dose, 1–10 μg/ml, also inhibited the incorporation of [ 3H]leucine into total protein. The reduced enzyme activity may therefore not be due to a direct effect of tunicamycin on the glycosylation of the lysyl oxidase protein itself but may be mediated through other actions of the drug. Colchicine caused no inhibition in lysyl oxidase activity secretion even at a 10 μM concentration, although it has been reported to inhibit collagen secretion at doses more than one order of magnitude lower.

Biochemical Defect in Chick Lung Resulting from Copper Deficiency

Lung tissue from groups of chicks raised 23 d on diets deficient in copper had a higher proportion of 0.15 M NaCl-extractable collagen but lower aldehyde-reacting compounds and elastin than controls the same age. Although the lungs from deficient chicks weighed less, the ratio of lung to body weight for the two groups stayed relatively constant throughout the 23-d period. Lysyl oxidase activity in lungs of deficient chicks was strongly suppressed. Feeding the deficient chicks the diet with copper supplements restored lysyl oxidase activity in lung. This showed the close and specific control of lung lysyl oxidase by copper. The impairment in cross-linking through lysyl oxidase failure is implied from these studies. Clearly, copper is needed for the normal development of lung.

Effect of prostaglandins on collagen synthesis in rabbit ovarian follicles during the ovulatory process

Collagen fibers in the ovarian follicle undergo drastic changes at ovulation due to the preovulatory increase of collagenolytic activities. The collagen synthesis in ovaries, however, has not been elucidated yet. To clarify the regulatory role of prostaglandins (PGs) in collagen synthesis of the follicular wall in relation to the ovulatory process, we measured prolyl hydroxylase (PH), as well as lysyl oxidase (LO) activity and the content of hydroxyproline (Hyp) in ovarian follicles of the rabbits treated by hCG, hCG/indomethacin (IM) and hCG/IM/various PGs. The experimental groups consisted of; 1) untreat control group 2) ovulatory group receiving hCG 3) non-ovulatory group given PGs 4) ovulatory group given hCG and PGs 5) group in which hCG-induced ovulation was inhibited by IM (4 mg/kg) 6) group in which IM-inhibited ovulation was recovered by PGF2 alpha (1.5 mg/kg) 7) group in which IM-inhibited ovulation was not restored by PGE1 (0.1 mg/kg) and PGE2 (0.7 mg/kg). The peak activities of PH and LO in ovarian follicles were observed at 12-13 hr after hCG injection, namely, immediately after ovulation. Significant changes of these activities after hCG administration were specific to the ovaries. PH activity in the ovaries was suppressed by the administration of IM, but LO activity was not significantly suppressed. In the hCG/IM/PGF2 alpha-treated ovulatory rabbits (Group 6), PH activity recovered to nearly the level of the hCG-treated rabbits (Group 2). By addition of PGE2, ovulation did not recover but PH activity was restored to about 70% of the hCG-treated rabbits. PGE1 did not have any effect on the reversal of ovulation-blockage or restoration of PH activity. The amount of Hyp after hCG administration tended to decrease from 6 hr to 10 hr but was significantly increased from 10 hr to 13 hr. This increase of Hyp after ovulation significantly correlated with the increase of PH and LO activities. In the hCG/IM/PGF2 alpha-treated rabbits (Group 6), the changes of Hyp were similar to those the hCG-treated rabbits (Group 2). In conclusion, collagen synthetic activity, found to be regulated by PH and LO activities in the ovarian follicles, was activated after follicle rupture, resulting in reconstruction of collagen fibers, and PGs play an important role in the ovulatory process by modifying collagen synthesis.

Skin lysyl oxidase activity is not rate limiting for collagen crosslinking in the glucocorticoid-treated rat.

Lysyl oxidase activity in the skin of rats receiving triamcinolone diacetate (12 mg/kg) for three consecutive days was decreased by sixty-four percent as compared to control values. A decrease of lysyl oxidase activity was observed twelve hours after the initial glucocorticoid injection. The decreased lysyl oxidase activity was accompanied by a forty-nine percent decrease of acetic acid extractable collagen. There was also a forty-two percent decrease in the alpha/beta ratio of the acetic acid soluble skin collagen of glucocorticoid-treated animals. These data indicate that although skin lysyl oxidase activity is decreased by glucocorticoid treatment, the crosslinking of acid extracted collagen as measured by the alpha/beta ratio and collagen solubility is increased. Accordingly lysyl oxidase activity is not rate limiting for collagen crosslink formation in the skins of rats treated with glucocorticoids.

Lysyl-oxidase activity in the odontoblast-predentine layer isolated from bovine incisor teeth

This layer had lysyl-oxidase (EC 1.4.3.13) activity, 4–11 times higher than either the sub-odontoblast layer or central pulp tissue, and similar to that in chick aorta, one of the tissues richest in such activity. This finding relates to the potential for rapid cross-link formation following synthesis of the predentine collagenous matrix.

Lung collagen and elastin after ozone exposure in vitamin B-6-deficient rats

The effects of vitamin B-6 deficiency and ozone exposure on selected features of connective tissue metabolism in lung were investigated in groups of weanling male rats fed one of three diets: (1) B-6-supplemented, fed ad lib; (2) B-6-deficient, fed ad lib; or B-6-supplemented, restricted to the food intake of deficient rats for 5 weeks. Also, perinatal rat pups were studied that were nursed from dams fed one of the 3 diets from parturition to day 15 of lactation. During the final week of each experiment, half of the rats in each of the groups were exposed to 0.64 ppm of ozone (23.5 h per day). The collagen and elastin content, collagen synthesis rate, total protein synthesis rate, and lysyloxidase activity of lungs were measured. Perinatal pups rendered vitamin B-6-deficient were particularly sensitive to ozone exposure (65% died as compared to fewer than 5% of the ad lib or food-restricted controls). When l-proline incorporation into collagen and total protein was investigated using lung minces, food restriction and B-6- deficiency resulted in about one-half the incorporation normally observed. Total lung lysyl oxidase activity was also decreased in B-6-deficient and food-restricted rats compared to B-6-supplemented rats fed ad lib. Exposure to ozone resulted in increased lysyl oxidase activity and collagen synthesis in lungs from B-6-supplemented rats, but such responses were not observed in B-6-deficient or foodrestricted (FR) rats exposed to ozone.

Evidence of an estrogen-like effect of dehydroepiandrosterone on lysyl oxidase activity in the mouse cervix.

The effect of dehydroepiandrosterone (DHA) on the lysyl oxidase activity in the mouse cervix was studied. Injection of animals ovariectomized 10 days before with 1 mg DHA was followed 24 h later by an increase in enzyme activity, which remained unaltered for 48 h, returning to control values by 96 h after injection. At least 100 micrograms of DHA was required to increase enzyme activity; the maximum effect occurred with 1 mg of the steroid and was comparable to that of 1 microgram estradiol-17 beta. These results demonstrate an estrogen-like effect of DHA on lysyl oxidase activity in mouse cervix. The onset of response was later and the duration of response was longer for DHA than for estradiol-17 beta.